By their nature, organisms contain many complex molecules and molecular assemblies. Some of the most important molecules and assemblies, including DNA, have high aspect ratios (i.e. one axis significantly greater in length than any other). It is known to use an optical apparatus to specifically detect these high aspect ratio molecules. Such an apparatus relies on the way these long molecules interact with polarised light (i.e. light with an electric field established in one direction only).
The phenomenon being exploited in the above apparatus is known as dichroism. The incident light may be either linearly polarised, giving rise to linear dichroism (LD), or circularly polarised, giving rise to circular dichroism (CD). LD is the property exhibited by some molecular structures whereby linearly polarised light is differentially absorbed along two orthogonal axes. CD relates to the difference in absorption of left and right circularly polarised light. A molecule that is capable of selective light absorption is known as a chromophore. Dichroic molecules, i.e. those that exhibit dichroic properties, are a particular type of chromophore. Examples of dichroic materials are certain natural crystals, stretched polymers, and other non-isotropic molecules. Biomolecules contain a wide range of chromophores (including aromatic side chains, nucleotides and peptide backbones).
In order to be able to observe a dichroic effect, it is necessary that the chromophores be aligned, or at least partially aligned, with respect to the incident polarised light beams. Some examples of moieties of interest that have been successfully aligned include linear biomolecules in the form of DNA, fibrous proteins and membranes (including membrane proteins) (Marrington R, Small E, Rodger A, Dafforn T R, Addinall S G, “FtsZ fiber bundling is triggered by a conformational change in bound GTP” J Biol Chem 2004; 279(47):48821-48829; Dafforn T R, Rajendra J, Halsall D J, Serpell L C, Rodger A, “Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couvette flow cell” Biophys J 2004; 86(1 Pt 1):404-410; Dafforn T R, Rodger A, “Linear dichroism of biomolecules: which way is up?” Curr Opin Struct Biol 2004; 14(5):541-546; Halsall D J, Rodger A, Dafforn T R, “Linear dichroism for the detection of single base pair mutations” Chem Commun (Camb) 2001(23):2410-2411).
A particularly convenient method for aligning such molecules is to create a solution including the molecules and then to flow the solution. Due to the elongate nature of the molecules, alignment arises as a result of shear forces generated by the flow, making the sample suitable for exhibiting the effect of linear dichroism.
In a known apparatus, once the molecules of interest have been aligned, linearly polarised light is directed through the solution from a direction substantially perpendicular to the axes of the aligned molecules. Absorption of light occurs within a molecule because, at a particular wavelength, the electric field of radiation urges the electrons in the molecule in a particular direction. When several molecules are similarly aligned, the electrons in each are all characterised by the same preferred net displacement direction. LD is a measure of the difference of absorbance of the incident light between two orthogonal polarisations. Varying the wavelength of the incident light and detecting the light emerging from the sample, allows a spectrum to be obtained which illustrates the absorbance of the sample with respect to wavelength.
An LD spectrum of a molecule provides information on the chromophores that are present including the orientation of the chromophores (and hence molecular conformation) and the orientation of the chromophores with respect to the axes of polarization. This information is important in understanding the structure of the molecule. Note that LD is a measurement of a sample's bulk property. The strength of the absorbance can be used to quantify the number of target molecules that are present in the sample. In addition, since LD is extremely sensitive to changes in alignment, an anomaly in the structure of a molecule may be detected. For example, LD can detect the distortion caused by a single mismatched hydrogen bond in a 1300 bp (base pair) fragment of DNA.
Furthermore, LD is extremely sensitive to the formation of a complex since the binding of an aligned molecule to a second molecule has the following two measurable effects:    1) The shape of the aligning moiety is altered and this results in its alignment also being altered, which leads to a change in the observed LD spectrum.    2) The second molecule itself becomes aligned by virtue of its attachment to the aligned molecule. This leads to the generation of an LD signal for the previously unaligned chromophores of the second molecule. Thus, information on the structure of the complex can be obtained.
Both of the above effects result in detectable phenomena that can be used to detect the formation of complexes. Not only can structural information be gleamed regarding the nature of the complex but the affinity of the interaction can also be determined.
There are many areas in which it is desirable to detect the presence of a specific of nucleic acid sequence. For example, in the detection of disease or bio-markers for particular genes the nucleic acid sequence can be used to detect the presence of a pathogen or a gene. In addition, the amount of the specific sequence that is present can be quantified.
One example of a method to detect small amounts of DNA is the polymerase chain reaction (PCR) in which DNA is amplified by a replication process using an enzyme and suitable substrates. The original double stranded sequence (target), if present, is separated into two single strands by the use of heat. Each single stranded DNA is bound by a short (typically 10-30 base) oligonucleotide (primer). The DNA is replicated by a (heat-stable) DNA polymerase enzyme using the target to determine the sequence and the primer as a starting point to give a new DNA molecule (amplimer). This process is repeated through several cycles to give exponential growth of the amplimer concentration. The building blocks are nucleotides.
The detection of the amplimer has previously been carried out in different ways, the main methods being:
Detection only at the end of the PCR:    1) Electrophoretic separation of the reaction components at the end of the reaction to detect the presence or absence of the nucleic acid target sequence
In order to detect the increasing amounts of the product during the reaction (q-PCR) and possibly use this to infer the starting concentration of the target:    2) Use of a dye that binds to only double-stranded nucleic acids but not to single stranded    3) Use of a dye that changes its fluorescence when bound to double-stranded nucleic acid    4) Labelling the oligonucleotide primers that are used in the amplification such that there are two dye molecules on opposite ends of the oligonucleotide that quench a fluorescence signal in the free form but give a large signal when bound to their complementary sequence.
As mentioned above, it is possible to detect DNA directly from its LD signal. In this approach the amplimer is detected directly, without the use of dyes, by virtue of its ability to align in shear flow in solution to a greater extent than the primers or nucleotides. The alignment is induced either by flow through a thin tube (capillary) or by Couette flow. The latter is achieved by the use of two coaxial cylinders (one inside the other) with an annular gap between them. One cylinder is rotated relative to the other and a shear gradient is formed causing some molecules that have one axis much longer than the other to align. In samples where there is a lot of amplimer the difference in absorbance will be large giving a large LD signal. In addition to being able to detect the amplimer at different cycle numbers, recently, through technical advances in the speed of heating and cooling the LD cells, it has become possible to carry out the amplification reaction in the LD alignment cell and to perform q-PCR. However, this approach of detecting the amplimer directly has two large disadvantages:                1) The amplimer must be long (>around 400 base pairs)        2) There is no scope for multiplexing (see below)        
WO 2008/059280 discloses a molecular sensor in which the sensor element comprised a scaffold moiety with a high aspect ratio having a receptor moiety attached thereto. The use of an alignable scaffold moiety as a substrate for the attachment of a receptor moiety meant that neither the receptor moiety itself nor the target molecule required inherent alignment properties. As well as being able to identify the aligned molecules through the resulting dichroic spectrum, the sensor can be used to quantify the aligned molecules and to detect the presence of molecular anomalies such as mismatches. The binding properties of the receptor moiety and target molecule may also be studied using the sensor. The inherent nature of dichroic molecules means that the sensor is extremely sensitive.
The present invention represents a further development of the sensor disclosed in WO 2008/059280 and aims to improve the application of dichroic analysis to nucleic acid molecules.